The melanocortin 3 receptor (MC3R) is involved in modulation of appetite, food intake, and energy expenditure in humans; therefore playing a potentially pivotal role in metabolic function. We found two missense MC3R sequence variants (C17A+G241A)) associated with obesity phenotypes that include increased body mass index (BMI), and increased adipose mass but with decreased lean mass in both pediatric patients and mouse models. In order to characterize the potential effects of human MC3R variants, humanized knock-in mouse models containing either WT or double mutant human MC3R variants (MC3RhWT/hWT or MC3RhDM/hDM, respectively) were generated in Dr. Yanovski's lab. These mice demonstrate an obese phenotype of greatly increased fat mass and decreased fat-free mass; however the mechanism through which human MC3R variants induce high adiposity remains unclear. Autophagy, a lysosome- dependent degradation pathway in eukaryotic cells, facilitates efficient nutrient redistribution processes when lipid droplets become substrates to efficiently remobilize the stored lipid molecules. The preliminary data in the proposed study suggest that function-decreasing MC3R mutations are strongly associated with down-regulated expression of transcription factor EB (TFEB, the master regulator of lysosomal lipid metabolism and autophagy) in both hepatic tissues and adipocytes from MC3RhDM/hDM compared with MC3RhWT/hWT. The long-term goal is to understand how MC3R signaling can be manipulated as possible therapeutic targets of obesity in children with MC3R polymorphisms. The objective is to determine if MC3R signaling affects autophagic regulation of lipid clearance in metabolically active tissues. The hypothesis is that MC3R plays an important role in the regulation of autophagy-mediated lipid metabolism in human and mouse obesity. The hypothesis will be tested by pursuing two specific aims: 1) to identify roles of MC3R as a modulator of autophagy pathway in fat metabolizing tissues from MC3RhWT/hWT and MC3RhDM/hDM, or MC3R-/- mice using microarray and protein analysis; and 2) to investigate molecular mechanisms by which hypoactive MC3R signaling perturbs autophagy- mediated lipid catabolism, using GFP-LC3 transgenic mice that contain humanized MC3R polymorphisms or null mutation. The proposed study is significant, because it is expected to uncover a novel mechanism of MC3R that is associated with childhood obesity. These investigations will ultimately allow us to explore the pathophysiological roles of human MC3R, providing important therapeutic interventions as well as advances in the field.